1. Field of the Invention
This invention relates to immunoassay procedures for quantitative and qualitative determination of antigenic materials in body fluids or cells and, more particularly, to a simple, precise colorimetric procedure for this analysis.
2. Description of the Prior Art
The quantitation of the level of various analytes in human serum or other body fluids is an essential means of diagnosis in certain disease states, and is widely used in many clinical situations. It is important that the tests used for such determination possess the maximum possible sensitivity and freedom from interference.
For example, in the case of certain proteins, quantitation of the amount present must rely on special properties of such proteins. Two examples of this are fibrinogen, with the special property of forming solid masses (clots), and enzymes, whose special property is facilitation and catalysis of metabolic reactions in vivo. In each case, present techniques of measurement rely upon indirect observation of the protein's special property rather than by direct quantitation of the protein per se.
In the case of the fibrinogen, a material whose function is the initiation of the clotting process, such as thrombin, is added to the sample, and the reaction allowed to proceed to completion. The amount of fibrinogen present is measured by protein determinations on the sample before and after such reaction, the clottable protein (fibrinogen) being expressed as the difference in protein content before and after the clot is formed and removed from the solution.
Similarly, in the case of enzyme tests, the present means of performing quantitative determinations rely on indirect means in which the enzyme is introduced into a premixed system, and its catalytic effect on the system is followed and quantified. No direct measurement of the amount of enzyme is provided for or is possible. Thus, any change in the system used for the test, i.e., the "substrate," may alter the change which the enzyme being measured produces, and, therefore, the value of the resulting measurement. In addition, other materials present in the sample being tested other than the enzyme may also cause a change in the test system. These interfering effects may be indistinguishable from the effect of the enzyme, and the error thus is introduced into the determination.
It would be desirable to measure the amount of materials such as those outlined above present in the sample directly rather than by indirect means. Such a direct measurement, however, requires extreme sensitivity since the amount of such materials present is usually very small. In the case of enzyme testing, the fact is compensated for in the present indirect methods by allowing the catalytic action to continue for varying periods of time. These lengthy procedures, however, introduce another opportunity for variation in result due to the change in test conditions, such as temperature, as the test is run.
The required sensitivity may be found in immunological test procedures, which are capable of detecting the presence of materials in minute quantities when an antibody to the material to be assayed has been produced, purified, and is then used as a reagent for measurement of the material in question. Immunoassay procedures currently employ radioactive isotopes bound to an antibody by measurement of the isotope level remaining after reaction with an antigen. An enzyme bound to an antibody is also employed in a similar fashion by measurement of conventional substrate absorbance changes at ultraviolet wavelengths. The third common means of measurement is by following the change in turbidity when antibody is added to a solution of antigen. This method is probably the least precise of all. Most conventional immunological tests rely for their quantitation on the reaction of the test system in a series of dilutions, the results being reported as the highest dilution (smallest concentration) of the material being analyzed which is capable of producing a "positive" reaction with the reagent (antibody). This method of measurement is deficient where more precise quantitation is desired. Moreover, all the methods currently employed utilize equipment which is inconvenient or frequently unavailable in routine laboratories.
Thus, it would be a significant improvement in the performance of such tests if the sensitivity of the immunological determinations could be applied to measurement of substances such as those set forth above in such a way that precise results could be obtained by direct measurement with devices ordinarily available in the routine laboratory.